Dissecting the key recognition features of the MS2 bacteriophage translational repression complex.
The MS2 RNA operator capsid offers an unparalleled opportunity to study sequence-specific protein-protein and RNA-protein interactions in molecular detail. RNA molecules encompassing the minimal translational operator recognition elements can be soaked into crystals of RNA-free coat protein shells, allowing the RNA to access the interior of the capsids and make contact with the operator binding sites. Correct interpretation of these structural studies depends critically on functional analysis in solution to confirm that the interactions seen in the crystal are not an artefact of the unusual approach used to generate the RNA-protein complexes. Here we present a series of in vivo and in vitro functional assays, using coat proteins carrying single amino acid substitutions at residues which either interact with the operator RNA or are involved in stabilizing the conformation of the FG loop, the site of the major quasi-equivalent conformational change. Variant operator RNAs have been assayed for coat protein affinity in vitro. The results reveal the robustness of the operator-coat protein interaction and the requirement for both halves of a protein dimer to contact RNA in order to achieve tight binding. They also suggest that there may be a direct link between the conformation of the FG loop and RNA binding.
Lago
H
H
School of Biology, University of Leeds, Leeds LS2 9JT, UK.
Fonseca
S A
SA
Murray
J B
JB
Stonehouse
N J
NJ
Stockley
P G
PG
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins; D019281:Dimerization; D014644:Genetic Variation; D017909:Levivirus / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D017354:Point Mutation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; D046911:Macromolecular Substances; D012367:RNA, Viral; D016601:RNA-Binding Proteins